In order to understand the mechanism of protein synthesis initiation, the required components will be purified by column chromatography and characterized by physical methods. To evaluate the initiation process, various combinations of components will be tested for their ability to interact with each other (i.e., the binding of met-tRNAf by eIF-2, the binding of mRNA by eIF-2, 4A or 4B, the binding of eIF-3 to 40S subunits). These interactions will be evaluated for influence by other initiation factors, nucleotide triphosphates, Mg ions, and inhibitors of protein synthesis initiation (such as aurin tricarboxylic acd, edeine, GDP). As an extension of these studies, mixtures containing all but one or two components will be analyzed (by sucrose density gradients) in an attempt to identify intermediates in the pathway to the formation of 80S initiation complexes. Previous studies have used primarily AUG codon; future experiments will use globin mRNA as a template. In particular, the requirement for ATP in the binding of mRNA to 40S subunits (reported by other laboratories) will be closely examined in an effort to define the component(s) responsible for the binding (and subsequent hydrolysis) of ATP.